The present invention relates to a heat shock factor (HSF) activity inhibitor, an expression suppressor for a substance encoded by a structural gene having a heat shock element (HSE) sequence in the gene region for transcriptional regulation, a treatment for or prevention of cancer based on the same, and a drug for treating or preventing stress diseases.
In order to produce a protein, a cell transcribes an mRNA from a DNA, i.e., a gene, and further translates the mRNA into the protein. It is clear that if the transcription is suppressed, the protein encoded by the DNA cannot be expressed. Transcription involves many factors called transcription factors. Transcription factors can be classified into basal transcription factors and transcription regulatory factors. While basal transcription factors are involved in most gene transcriptions, transcription regulatory factors are involved in transcriptions during a particular period or under particular conditions. HSF is one of such transcription regulatory factors, and is present in an evolutionarily wide range of species, from yeast to humans. HSF is characterized in that it specifically induces expression of a group of genes controlled thereby although transcription or protein synthesis in general is suppressed under environmental or physical stress such as heat or chemicals such as an amino acid analog, ethanol or a peroxide. It is known that upon activation HSF is turned from a monomer into a homotrimer in the cytoplasm and is transferred into the nucleus so as to recognize and bind to a binding sequence on a DNA called the HSE sequence (a base sequence of about 8 bp or more), thereby inducing and transcribing an mRNA (Mol. Cell. Biol., 13: 2486, 1993). Four types of HSF, HSF1 to HSF4, have been reported. It is believed that HSF1 is induced by external stress such as heat (Mol. Cell. Biol., 17: 469, 1997). Proteins which have the HSE sequence and are transcribed and translated from genes controlled by HSF are generically called heat shock proteins (HSPs), stress proteins, or chaperone proteins. Examples of HSPs include, for example, HSP70, HSP90, HSP100, etc., and it has been reported that HSF actually functions in the expression of these HSPs. Conventionally, flavonoids such as quercetin are known as HSF inhibitors (Cell Struct. Func., 15: 393, 1990).
Stress includes cellular-level stress and animal-level stress, and the relationship between the cellular-level regulation/control of stress and the animal-level treatment of stress is an issue that should be studied. The present invention provides treatment of diseases by controlling the expression of a protein which is induced by stress, i.e., by HSF. Many HSPs are usually synthesized in a cell through a transcription mechanism other than the HSF activation. Some HSPs are believed to be essential for the cell to function properly (Nature, 355: 33, 1992; Nature, 381: 571, 1996). Generally, an inhibitor which provides its function by binding to a protein may, depending on the type of the target protein, affect cells or tissues which are not intended to be treated, thereby causing serious side effects. Since HSF is a stress-induced transcription factor, its inhibitor is believed to inhibit the transcription or the signal transmission during the HSF activation. By nature, HSF is activated to induce expression only when the cell is under a specific environment. The HSF inhibition as used herein aims to affect as little as possible the expression of the essential HSPs which are normally required by the cell while suppressing only the induction by stress, including abnormal induction. Heretofore, there has not been any drug which is capable of specifically suppressing only the induction under stress such as heat, and yet satisfactory in terms of the effectiveness, specificity, or the like.
The HSF activation process, i.e., the HSF modification and signal transmission caused by a stress stimulus such as heat being applied to the cell, has not yet been sufficiently elucidated. If a specific inhibitor is discovered, because of its mechanism it is expected to have a potential to be a new type drug. For example, it is known that a cell acquires a resistance to heat for a certain period of time after a thermotherapy for cancer. This is called acquisition of heat resistance. Since it takes 4-5 days for the heat resistance to disappear, a thermotherapy is typically conducted with a frequency of once or twice per week. It is expected that, if the acquisition of heat resistance can be disabled, the effect of cancer thermotherapy can be improved because it will then be possible to increase the number of times the therapy can be conducted. Moreover, it is clinically widely recognized that the effect of thermotherapy for cancer is greater when it is performed in combination with radiotherapy, chemotherapy, or the like, than when it is performed alone. For the mechanism of acquiring the heat resistance, it is believed that, for example, a protein which has been or is being denatured, is recovered by the action of HSP inducibly produced in a cell by heat, thereby reducing the cell killing effect. However, the details are still unclear. It is believed that HSP prevents apoptosis in a cancer cell. Therefore, it is possible, by suppressing the expression and induction of HSP, to cause normal apoptosis in a cancer cell, thereby controlling immortality or infinite proliferation of the cancer cell.
In the modern world, there has been an increasing number of cases of stress diseases caused by mental stress, including, for example, depression and anxiety. Many of the conventional drugs therefor have not been satisfactory in terms of their effectiveness and side effects. Moreover, the conventional drugs take a long time before they exhibit their effects, and the mechanism of action cannot be explained only with the receptor theory. Under such circumstances, a drug which selectively inhibits reuptake of serotonin has attracted attention as an antidepressant with reduced side effects. On the other hand, a serotonin (5-HT) receptor agonist has also attracted attention as an antianxiety drug. Some of these drugs (e.g., tandospirone citrate) provide both an antidepression action and an antianxiety action. This suggests that depression and anxiety, as stress diseases, are controlled at least partially by a common mechanism. It is expected that the essential characteristics of stress diseases such as depression and anxiety are elucidated, and a new drug are then developed based on a novel action mechanism. One strategy therefor is to associate the cellular-level stress with the animal-level stress from a novel point of view. The present inventors assumed that a drug which is capable of inhibiting activation of a heat shock factor at cellular level, would have an effect on stress diseases such as depression and anxiety at an animal level. Stress and a response thereto have a fundamental structure which is common among hierarchically different levels, i.e., the cell and the animal (Ichiro YAHARA, Stress Protein (ed., Kazuhiro NAGATA) pp. 257-262, 1994). This is supported by following: it is recognized that a stress response is purposive in that exposure to a stress make an animal or cell resistant to the next stress; a stress resistance is similarly obtained through different types of stress; stress is normally transient in an animal or cell, and the like. In view of these characteristics of stress, the present invention aims to treat a disease by controlling an overreaction or uncontrolled reaction in the individual cells, thereby restoring it to a normal state. The inhibition of signal transmission of the heat shock factor activation may be believed to inhibit the stress reaction of a cell and to place the cell in a stress-free state. Therefore, the suppression of stress reaction at a cellular-level through suppression of the HSF activation or the HSP expression can lead to suppression of stress reaction at an animal-level, whereby the inhibition of HSF activity or HSP expression can result in treatment or prevention of stress diseases such as depression and anxiety.
Currently, flavonoids such as quercetin is believed to have suppressive action on the HSP production, and to inhibit the activation of HSF by suppressing the expression of its mRNA. However, the mechanism of the action has not completely been known. Quercetin does not have a sufficient inhibitory effect on the HSF activity for use as a therapeutic. Moreover, there is fear of quercetin exhibiting potential side effects since it has various other physiological activities such as mutagenicity, an antivirus activity, a tyrosine kinase inhibiting activity, a protein kinase C inhibiting activity, and a lactate transport inhibiting activity.
Therefore, it has been desired for a drug to be developed which acts on the HSF activation more specifically so as to have a stronger effect and reduced side effects.
The present inventors have conducted extensive research in view of the above-described circumstances. As a result, the present inventors have discovered a drug which is effective in inhibiting the acquisition by a cell of heat resistance, such as that occurring in thermotherapy for cancer, through the inhibitory action on the activity of HSF as a transcription regulatory factor. Moreover, the present inventors have found, as the HSF activity inhibitor showed an antidepression action, etc., that it is also effective for stress diseases at animal level as well as cellular-level stress, thereby completing the present invention.
According to the first aspect, the present invention relates to a compound represented by general formula (I): 
wherein X is xe2x80x94CH2xe2x80x94 and R1 is C1-C2 alkyl, formyl or halogen; or X is xe2x80x94(CH2)2xe2x80x94 and R1 is C1-C3 alkyl, formyl, acetyl, hydrogen or halogen. In general formula (I), the olefinic double bond may take either an E-configuration or a Z-configuration.
In a preferred embodiment, the present invention relates to a compound wherein X is xe2x80x94CH2xe2x80x94 and R1 is formyl.
In another preferred embodiment, the present invention relates to a compound wherein X is xe2x80x94(CH2)2xe2x80x94 and R1 is hydrogen.
In still another preferred embodiment, the present invention relates to a compound wherein X is xe2x80x94(CH2)2xe2x80x94 and R1 is formyl.
According to the second aspect, the present invention relates to an inhibitor of heat shock factor activity comprising, as an active ingredient thereof, a compound represented by general formula (I): 
wherein X is xe2x80x94CH2xe2x80x94 and R1 is C1-C2 alkyl, formyl, acetyl, hydrogen or halogen; or X is xe2x80x94(CH2)2xe2x80x94 and R1 is C1-C3 alkyl, formyl, acetyl, hydrogen or halogen.
In a preferred embodiment, the heat shock factor is HSF1.
In a preferred embodiment, the active ingredient is represented by general formula (I) wherein X is xe2x80x94CH2xe2x80x94 and R1 is formyl.
In another preferred embodiment, the active ingredient is represented by general formula (I) wherein X is xe2x80x94(CH2)2xe2x80x94 and R1 is hydrogen.
In still another preferred embodiment, the active ingredient is represented by general formula (I) wherein X is xe2x80x94CH2xe2x80x94 and R1 is hydrogen.
In still another preferred embodiment, the active ingredient is represented by general formula (I) wherein X is xe2x80x94CH2xe2x80x94 and R1 is acetyl.
According to the third aspect, the present invention relates to an expression suppressor for a substance encoded by a structural gene having a heat shock element sequence in the gene region for transcriptional regulation, the suppressor comprising, as an active ingredient thereof, a compound represented by general formula (I): 
wherein X is xe2x80x94CH2xe2x80x94 and R1 is C1-C2 alkyl, formyl, acetyl, hydrogen or halogen; or X is xe2x80x94(CH2)2xe2x80x94 and R1 is C1-C3 alkyl, formyl, acetyl, hydrogen or halogen.
In a preferred embodiment, the substance encoded by the structural gene having the heat shock element sequence in the gene region for transcriptional regulation is selected from the group consisting of HSP40, HSP47, HSP70, HSP90, HSP100, IL-1, xcex1-fetoprotein, IFN-xcex1, vitellogenin, and P-glycoprotein.
According to the fourth aspect, the present invention relates to a drug for treating or preventing a disease for which an inhibitory action on heat shock factor activity, or a suppressing action on the expression of a substance encoded by a structural gene having a heat shock element sequence in the gene region for transcriptional regulation is effective, the drug comprising, as an active ingredient thereof, a compound represented by general formula (I): 
wherein X is xe2x80x94CH2xe2x80x94 and R1 is C1-C2 alkyl, formyl, acetyl, hydrogen or halogen; or X is xe2x80x94(CH2)2xe2x80x94 and R1 is C1-C3 alkyl, formyl, acetyl, hydrogen or halogen.
In a preferred embodiment, the disease for which an inhibitory action on heat shock factor activity, or the suppression action on the expression of the substance encoded by the structural gene having the heat shock element sequence in the gene region for transcriptional regulation is effective is cancer.
According to a more preferred embodiment, in general formula (I), X is xe2x80x94CH2xe2x80x94 and R1 is formyl.
In another preferred embodiment, the disease for which an inhibitory action on heat shock factor activity, or the suppressing action on the expression of the substance encoded by the structural gene having the heat shock element sequence in the gene region for transcriptional regulation is effective is a stress disease.
In a more preferred embodiment, the stress disease is depression.
According to a preferred embodiment, in general formula (I), X is xe2x80x94(CH2)2xe2x80x94 and R1 is hydrogen, or X is xe2x80x94CH2xe2x80x94 and R1 is hydrogen.
According to the fifth aspect, the present invention relates to a drug for treating or preventing a stress disease which exhibits an inhibitory action on heat shock factor activity, or a suppressing action on the expression of a substance encoded by a structural gene having a heat shock element sequence in the gene region for transcriptional regulation.
The present invention relates to a novel compound represented by general formula (I): 
wherein X is xe2x80x94CH2xe2x80x94 and R1 is C1-C2 alkyl, formyl or halogen; or X is xe2x80x94(CH2)2xe2x80x94 and R1 is C1-C3 alkyl, formyl, acetyl, hydrogen or halogen.
In general formula (I), the olefinic double bond may take either an E-configuration or a Z-configuration.
A preferred embodiment of the present invention is a compound represented by structural formula (I): 
wherein X is xe2x80x94CH2xe2x80x94 and R1 is formyl.
Another preferred embodiment of the present invention is a compound represented by structural formula (II): 
wherein X is xe2x80x94(CH2)2xe2x80x94 and R1 is hydrogen.
Regarding a method for producing a compound of the present invention represented by general formula (I): 
wherein X is xe2x80x94CH2xe2x80x94 and R1 is C1-C2 alkyl, formyl, acetyl, hydrogen or halogen; or X is xe2x80x94(CH2)2xe2x80x94 and R1 is C1-C3 alkyl, formyl, acetyl, hydrogen or halogen, the compound can be obtained by reacting piperonal as shown below: 
with a compound represented by general formula (II): 
wherein X is xe2x80x94CH2xe2x80x94 and R1 is C1-C2 alkyl, formyl, acetyl, hydrogen or halogen; or X is xe2x80x94(CH2)2xe2x80x94 and R1 is C1-C3 alkyl, formyl, acetyl, hydrogen or halogen, in the presence of a basic catalyst (e.g., an alkaline metal hydride such as sodium hydride or potassium hydride, an alkaline metal alcoholate such as sodium methylate or sodium ethylate, an alkaline metal amide such as lithium diisopropylamide, an organic acid salt such as sodium acetate, an alkaline metal carbonate such as sodium carbonate or potassium carbonate, an alkaline metal hydroxide such as sodium hydroxide, an organic acid base such as piperidine, etc.).
Alternatively, the compound can be obtained by reacting a compound represented by general formula (III): 
wherein X is xe2x80x94CH2xe2x80x94 and R1 is C1-C2 alkyl, formyl, acetyl, hydrogen or halogen; or X is xe2x80x94(CH2)2xe2x80x94 and R1 is C1-C3 alkyl, formyl, acetyl, hydrogen or halogen, with a halogeno alkane such as iodomethane, an organic acid such as formic acid, an organic acid halide such as acetyl chloride, or a halogen such as chlorine, through a condensation reaction such as dehydrogenation condensation, or dehydrohalogenation condensation.
The present invention provides an inhibitor of heat shock factor activity, an expression suppressor for a substance encoded by a structural gene having a heat shock element sequence in the gene region for transcriptional regulation, or a drug for treating or preventing a disease for which an inhibitory action on heat shock factor activity or a suppressing action on the expression of a substance encoded by a structural gene having a heat shock element sequence in the gene region for transcriptional regulation is effective, the inhibitor or the suppressor comprising, as an active ingredient thereof, a compound represented by general formula (I): 
wherein X is xe2x80x94CH2xe2x80x94 and R1 is C1-C2 alkyl, formyl, acetyl, hydrogen or halogen; or X is xe2x80x94(CH2)2xe2x80x94 and R1 is C1-C3 alkyl, formyl, acetyl, hydrogen or halogen. The active ingredient can inhibit the activity of the transcriptional control factor HSF, thereby inhibiting induction of the transcription of a structural gene having a DNA sequence recognized by HSF (i.e., HSE) in the gene region for transcriptional regulation. Thus, the active ingredient specifically suppresses a protein induced/expressed by unusual stress such as heat while not inhibiting the basic expression of molecular species which are essential for the cell. Therefore, the active ingredient is capable of effectively inhibiting and suppressing a protein which is encoded by a structural gene having HSE in the gene region for transcriptional regulation and which is induced/expressed by stress such as heat.
For typical stress proteins including a small molecular HSP such as HSP27 or HSP47, an HSP60 family, an HSP70 family, an HSP90 family and an HSP100 family, a certain type of enzymes involved in saccharometabolism, IL-1, xcex1-fetoprotein, IFN-xcex1, vitellogenin, P-glycoprotein, or the like, for example, an HSE sequence recognized by HSF is present in the promoter sequence for the coded gene. Therefore, it is believed that suppressing the induction of the gene expression from such genes can result in preventing/treating diseases related to the genes. Therefore, a compound which exhibits inhibitory action on the heat shock factor activity or a suppressing action on the expression of a substance encoded by a structural gene having a heat shock element sequence in the gene region for transcriptional regulation is effective in treating and preventing, for example, cancer and a disease, such as depression or anxiety, which is considered to be caused by stress. Therapies for cancer include thermotherapy for cancer alone, thermotherapy for cancer in combination with radiotherapy or a chemotherapeutic, and resistance-overcoming therapy wherein the resistance acquired by a cancer cell is overcome through the inhibition of multidrug resistance acquisition, thereby recovering the effect of a chemotherapeutic on the cancer cell.
The inventive inhibitor of heat shock factor activity, or the inventive suppressor of the expression of a substance encoded by a structural gene having a heat shock element sequence in the gene region for transcriptional regulation, improves the therapeutic effect of thermotherapy for cancer based on its suppressing action on cellular heat resistance. The dosage of the compound of the present invention for a human in thermotherapy for cancer can be determined by a method known in the art. For example, the dosage for a human can be determined as a value equivalent to the dosage suitable for a mouse which is determined by a test as described in Example 8. The dosage for a mouse is typically in the range of 0.01 to 2000 mg/kg, preferably 0.1 to 1000 mg/kg, more preferably 1 to 500 mg/kg.
The inventive inhibitor of heat shock factor activity or the inventive suppressor of expression of a substance encoded by a structural gene having a heat shock element sequence in the gene region for transcriptional regulation, also exhibit an antidepression action. The dosage of the compound of the present invention as an antidepressant can be determined by a method well known in the art. For example, the dosage for a human can be determined as a value equivalent to the dosage suitable for a mouse which is determined by a test as described in Example 7. The dosage for a mouse is typically in the range of 0.1 to 5000 mg/kg, preferably 1 to 2000 mg/kg, more preferably 10 to 500 mg/kg. Other depression models from which an appropriate dosage can be derived include a forced swimming test as described in Example 7 as well as a stress (chronic stress) model, a muricide model, a reserpine antagonizing model, an electrically self-stimulation model (Psychopharmacology, 83: 1, 1984), a tail suspension model (Jpn. J. Psychopharmacol., 12: 207, 1992).
The effect on other stress diseases of the inventive inhibitor of the heat shock factor activity or the inventive suppressor of the expression of a substance encoded by a structural gene having a heat shock element sequence in the gene region for transcriptional regulation can be confirmed by pharmacological or behavioral pharmacological experiments that are well known in the art. For the antianxiety action, for example, a conflict model (Jpn. J. Psychopharmacol., 15: 115, 1995), an elevated plus maze method (Jpn. J. Psychopharmacol., 15: 125, 1995), a mental stress loading method (Jpn. J. Psychopharmacol., 15: 375, 1995), and the like, may be used. Experimental methods for other stress diseases are described in detail in, for example, xe2x80x9cDoubutsu moderu riyo shusei [Collection of Animal Model Usage]xe2x80x9d (Ryuta ITO, et al. eds., RandD Planning, 1985). The dosage of the compound of the present invention for a human can be determined by a method well known in the art. For example, the dosage for a human can be extrapolated from the effective dosage determined in the above-described animal experiments by using a method well known in the art.
The actual dosage is adjusted by a clinician according to the need of a particular patient.
The inventive inhibitor of the heat shock factor activity or the inventive suppressor of the expression of a substance encoded by a structural gene having a heat shock element sequence in the gene region for transcriptional regulation, or a drug comprising the same for treating or preventing cancer or stress diseases can be administered orally, enterally or parenterally. Specific types of formulation include, for example, an oral formulation such as tablet, powder, capsule, granule, fine granule or syrup, a parenteral formulation such as suppository, ointment, injection, cream for topical application or eye drop.
The inventive inhibitor of the heat shock factor activity or the inventive suppressor of the expression of a substance encoded by a structural gene having a heat shock element sequence in the gene region for transcriptional regulation, a drug comprising the same for treating or preventing cancer or a drug comprising the same for treating or preventing stress diseases can be formulated with a carrier which may optionally comprise any organic or inorganic, solid or liquid component suitable for oral, enteral or parenteral administration. For example, such an optional component may be a excipient such as crystalline cellulose, gelatin, lactose, saccharose, starch, corn starch, dextrin, mannite, magnesium stearate, talc, vegetable and animal fats, oil, gum, polyalkylene glycol, Arabian gum or pectin, a binder, a disintegrator, a lubricant, a flavor, a filler, a coloring agent, a stabilizer, an isotonic agent, a solubilizer, a dispersant, a solubilizing agent, an emulsifier, a coloring agent, a wetting agent, and an antioxidant. The compound of the present invention can be included in the formulation at 0.001 to 100%. Any other compatible inhibitor or drug may further be included. In such a case, the compound of the present invention is not necessarily the main component of the formulation.
Although he present invention will now be described in greater detail by way of examples, it is not limited to those examples.
The compound of the present invention can be synthesized as follows.